Recombinant Oenothera parviflora ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in Oenothera parviflora?

ATP synthase subunit c (atpH) in Oenothera parviflora is a small hydrophobic protein consisting of 81 amino acids that forms part of the F0 sector of the chloroplastic ATP synthase complex. The protein's amino acid sequence is: MNPLISAASVIAAGLAVGLASIGPGIGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV .

This protein functions as a key component of the c-ring of the ATP synthase, which is embedded in the thylakoid membrane and facilitates proton translocation. Multiple copies of subunit c assemble to form a ring structure that rotates during ATP synthesis, converting the proton motive force generated by photosynthetic electron transport into mechanical energy that drives ATP production.

The F-type ATP synthase in chloroplasts consists of two main parts: the membrane-embedded F0 sector (containing subunit c) and the soluble F1 sector. The c-ring in the F0 sector acts as a rotor that couples proton flow across the membrane to ATP synthesis in the F1 sector .

How does atpH vary among different Oenothera species?

Comparative genomic analyses reveal that ATP synthase components, including atpH, show notable sequence conservation among Oenothera species, though with subtle variations that may impact photosynthetic efficiency. When examining different Oenothera species and their hybridization patterns, ATP synthase function appears to be maintained even in certain incompatible plastid-nuclear combinations.

Importantly, when comparing Oenothera species with incompatible plastid-nuclear combinations that display photosynthetic deficiencies, researchers have observed that "the disturbance in acclimation response is independent of ATP synthase and PC function" , suggesting that atpH and other ATP synthase components maintain functional conservation despite broader photosynthetic incompatibilities.

What expression systems are optimal for recombinant production of Oenothera parviflora atpH?

Escherichia coli represents the preferred expression system for recombinant production of Oenothera parviflora atpH. Based on successful approaches with similar proteins, the following methodology is recommended:

Vector Selection:

• pMAL-c2x vectors containing maltose-binding protein (MBP) fusion constructs have demonstrated success for similar chloroplast proteins

• pET-32a(+) systems with thioredoxin fusion tags can enhance solubility

• pFLAG vectors may be utilized for immunodetection applications

Host Strain Considerations:
E. coli T7 Express lysY/Iq strains have proven effective, particularly when co-transformed with chaperone-expressing plasmids such as pOFXT7KJE3 (expressing DnaK, DnaJ, and GrpE chaperones), which "substantially increase quantities of recombinant proteins which are toxic or otherwise difficult to produce" .

Expression Protocol:

• Transform E. coli with the chosen expression vector

• Culture in LB-glucose medium (1.0% tryptone, 0.5% yeast extract, 0.4% glucose, 0.5% NaCl) with appropriate antibiotics

• Grow at 37°C with orbital shaking (200 RPM) until reaching OD600 of 0.6-0.7

• Induce expression with IPTG (optimal concentration determined empirically)

• Continue incubation under optimized temperature conditions

The selection of appropriate fusion tags is crucial for enhancing solubility and facilitating purification, as hydrophobic membrane proteins like atpH often form inclusion bodies when expressed in bacterial systems.

What purification strategies yield the highest purity and activity for recombinant atpH?

Purification of recombinant atpH requires specialized approaches due to its hydrophobic nature as a membrane protein. Based on successful methods for similar ATP synthase subunits, the following multi-step strategy is recommended:

Initial Extraction and Solubilization:

• Harvest bacterial cells via centrifugation and resuspend in appropriate buffer

• Disrupt cells via sonication or French press

• Isolate inclusion bodies or membrane fractions through differential centrifugation

• Solubilize membrane proteins using mild detergents (e.g., n-dodecyl β-D-maltoside) or denaturants (with subsequent refolding)

Affinity Chromatography:
For His-tagged constructs:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices

• Binding buffer containing appropriate detergent concentrations to maintain protein solubility

• Step-wise or gradient elution with increasing imidazole concentrations

Storage and Stability:

• Store purified protein at -20°C/-80°C

• Add 5-50% glycerol (final concentration) to enhance stability during freeze-thaw cycles

• Aliquot to avoid repeated freeze-thaw cycles

Reconstitution Procedure:
"Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL" for optimal stability and subsequent experimental applications.

This purification approach typically yields protein with "greater than 90% purity as determined by SDS-PAGE" , suitable for structural and functional studies.

How can recombinant atpH be used to study ATP synthase assembly and c-ring formation?

Recombinant atpH provides a valuable tool for investigating ATP synthase assembly and c-ring formation through the following methodological approaches:

In vitro Reconstitution Studies:
Purified recombinant atpH can be used for experiments focused on "the reconstitution of the multimeric ring (cn)" . This approach allows researchers to study:

• Factors influencing c-subunit oligomerization

• Determinants of c-ring stoichiometry

• Lipid requirements for proper assembly

Site-Directed Mutagenesis Applications:
The recombinant expression system enables "molecular biology techniques which cannot otherwise be applied to a native cn ring" , including:

• Targeted amino acid substitutions to identify residues critical for c-ring assembly

• Introduction of reporter groups (e.g., fluorescent or spin labels) at specific positions

• Creation of variants to probe proton-binding sites and translocation pathways

Hybrid Complex Formation:
Recombinant atpH can be combined with other ATP synthase components to study:

• Interactions between the c-ring and other F0 subunits

• Assembly with F1 components to form functional F0F1 complexes

• Species-specific compatibility between ATP synthase subunits

These approaches contribute to understanding "the stoichiometric variation of the intact ring" , a fundamental aspect of ATP synthase structure and function with implications for energy conversion efficiency in chloroplasts.

What biophysical techniques are most informative for analyzing recombinant atpH structure and function?

Multiple complementary biophysical techniques provide insights into the structure, dynamics, and function of recombinant atpH:

Structural Characterization:

• Circular Dichroism (CD) Spectroscopy: Quantifies secondary structure content (α-helices predominate in atpH)

• Nuclear Magnetic Resonance (NMR): Provides atomic-level structural information in membrane-mimetic environments

• X-ray Crystallography: When incorporated into c-rings, reveals precise structural arrangements

Functional Analysis:

• Reconstitution into Liposomes: Measures proton translocation activity

• Patch-Clamp Electrophysiology: Quantifies ion conductance through reconstituted channels

• ATP Synthesis Assays: Assesses functional integration with other ATP synthase components

Interaction Studies:

• Förster Resonance Energy Transfer (FRET): Monitors distances between labeled subunits

• Chemical Cross-linking: Identifies interaction interfaces between atpH and other subunits

• Surface Plasmon Resonance (SPR): Measures binding kinetics and affinities

These methodologies can be applied to investigate the specific properties of Oenothera parviflora atpH and compare them with homologs from other species, providing insights into structure-function relationships and evolutionary adaptations.

How can recombinant atpH contribute to understanding plastid-nuclear incompatibility in Oenothera hybrids?

Recombinant atpH provides powerful tools for investigating the molecular mechanisms underlying plastid-nuclear incompatibility in Oenothera hybrids through several experimental approaches:

Hybrid Protein Interaction Studies:
Recombinant atpH from different Oenothera species can be used to examine interactions with nuclear-encoded ATP synthase subunits from various genetic backgrounds, potentially revealing:

• Compatibility determinants at protein-protein interfaces

• Species-specific structural adaptations affecting complex assembly

• Compensatory mutations that maintain functional integration

Reconstitution with Mixed Subunit Origins:
By combining recombinant atpH from one Oenothera species with other ATP synthase components from different species, researchers can:

• Recreate incompatible combinations in controlled in vitro systems

• Identify specific interaction failures leading to dysfunction

• Test the functional consequences of natural sequence variations

Light Response Experiments:
Research has shown that incompatible plastid-nuclear combinations in Oenothera exhibit light-dependent phenotypes. While "the disturbance in acclimation response is independent of ATP synthase and PC function" in some hybrid combinations, detailed studies with recombinant atpH can further elucidate:

• How ATP synthase adjusts to changing light conditions in different genetic backgrounds

• Whether subtle functional differences in ATP synthase contribute to broader photosynthetic inefficiencies

• The relationship between ATP synthesis capacity and electron transport chain components under various light regimes

These approaches provide molecular-level insights into the broader observation that "chloroplast–nuclear incompatibility... usually manifests in bleached plants" in Oenothera hybrids, contributing to our understanding of speciation mechanisms.

What role does atpH play in photosynthetic acclimation to different light conditions in Oenothera species?

ATP synthase components, including atpH, appear to maintain functional stability across different light conditions in Oenothera species, even as other photosynthetic parameters show significant variation. Experimental findings support several key observations:

Plastome-Specific Responses:
The observed variations in photosynthetic parameters under high light conditions were primarily determined by the plastome type rather than the nuclear genome, as demonstrated by the similar responses of AA-II and AB-II genotypes compared to AA-I plants. This suggests that "plastome I is better adapted to cope with HL conditions than plastome II" .

ATP Synthase Stability:
While parameters like electron transport capacity, chlorophyll content, and chlorophyll a/b ratio showed marked changes in response to increased light intensity in some genotypes, chloroplast ATP synthase activity remained relatively stable, suggesting a degree of functional conservation and regulatory independence.

These findings indicate that atpH and the ATP synthase complex may maintain functional consistency across changing light conditions, even as other components of the photosynthetic apparatus undergo significant adjustments. This apparent stability makes recombinant atpH an interesting tool for comparative studies of photosynthetic adaptation mechanisms.

How does atpH sequence conservation compare to other chloroplast-encoded genes in Oenothera species?

Comparative genomic analyses reveal distinct patterns of sequence conservation across chloroplast genes in Oenothera species, providing context for understanding atpH evolution:

Variability Across Chloroplast Regions:
When examining nucleotide diversity across chloroplast genomic regions in Oenothera and other genera, researchers have identified regions of higher and lower variability. The following table presents the most variable regions in Oenothera compared to other plant genera:

ATP Synthase Genes in Context:
While the atpH gene specifically is not highlighted among the most variable regions in this dataset, it's noteworthy that photosynthesis-related genes show varying levels of conservation. For instance, the psbJ-psbL region (encoding Photosystem II proteins) shows moderate variability, ranking second among the most variable regions in Oenothera .

Functional Constraints:
The relative conservation levels can be interpreted in terms of functional constraints, with genes encoding core functional domains of essential proteins typically showing higher conservation. As a critical component of the ATP synthase complex, atpH would be expected to maintain high sequence conservation in regions essential for c-ring formation and proton translocation.

These patterns of sequence conservation provide evolutionary context for understanding the structural and functional properties of atpH in Oenothera species and their hybrids.

How can recombinant atpH be used to study the impact of environmental stressors on ATP synthase function?

Recombinant atpH provides a versatile experimental system for investigating how environmental stressors affect ATP synthase structure, assembly, and function in Oenothera species:

Oxidative Stress Studies:
Recombinant atpH can be exposed to controlled oxidative conditions to:

• Identify oxidation-sensitive residues through mass spectrometry

• Assess the impact of oxidative modifications on c-ring assembly

• Determine how oxidative damage affects proton translocation efficiency

Temperature Adaptation Experiments:
By subjecting recombinant atpH to varying temperature conditions, researchers can:

• Compare thermal stability of atpH from different Oenothera species

• Investigate temperature-dependent conformational changes

• Assess how temperature affects interactions with other ATP synthase subunits

Integration with Antioxidant Systems:
Oenothera species contain various bioactive compounds with antioxidant properties, including "gallic acid, caffeic acid, epicatechin, coumaric acid, ferulic acid, rutin and rosmarinic acid" . Studies combining recombinant atpH with these compounds can reveal:

• Potential protective effects of specific antioxidants on ATP synthase function

• Mechanisms by which plant antioxidant systems preserve bioenergetic efficiency

• Species-specific adaptations in stress response pathways

Experimental Design Considerations:
When using recombinant atpH for stress response studies, researchers should:

• Compare results between in vitro systems and intact chloroplasts

• Consider the impact of lipid environment on protein response to stressors

• Develop appropriate functional assays to quantify stress-induced changes in activity

These approaches expand our understanding of how photosynthetic energy conversion adapts to environmental challenges, with potential applications in improving crop resilience to changing climate conditions.